Our research is directed toward expanding the genetic tools available for the study of cultured mammalian cells and their viruses. We are developing the methodology for isolating nonsense mutants and their suppressors in mammalian cells. The system we have chosen will also permit a detailed analysis of the mechanisms of chemical mutagenesis and a genetic method for studying the mechanism of selective degradation of abnormal proteins in mammalian cells. Nonsense mutants would be particularly useful for studying the physiology of mammalian viruses because: a) they would exhibit a clear mutant phenotype (i.e., they should not be subject to "leakage" problems observed with temperature-sensitive mutants) and, b) they can be employed for directly identifying the mutant gene product. For this project, a large set of mammalian cell lines (500) lacking hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (less than 0.2% of wild type) have been isolated. Each of these cell lines has been screened for the presence or absence of immunologically cross-reacting material (CRM) to wild-type enzyme. The presence of a nonsense mutant of HGPRT among this collection of HGPRT cell lines will be determined by using a specific radioimmune assay combined with sub-unit molecular weight measurements on calibrated SDS-polyacrylamide gels. Revertants of the CRM cell lines will be assayed in vitro for the presence of suppressor tRNA activity. A nonsense mutant can revert either by direct back-mutation, or by acquiring a suppressor tRNA. We believe that the latter mechanism will be favored because of the greater number of potential mutable sites which could convert a normal tRNA into a suppressor tRNA.